Since the advent of molecular genetics the correction of human genetic lesions through the introduction of replacement genes has been a goal of human gene therapy. Various methods of gene replacement have been investigated including in situ replacement of a disease gene or portion thereof, supplementation of a disease gene through providing a replacement gene exogenously, e.g., on a non-integrating transgene or a transgene designed to integrate at a exogenous locus, and various other methods. While these approaches have appeared promising in the laboratory and even in some pre-clinical animal models, in human clinical trials they have been hampered by many roadblocks including induction of a host immune activity in response to the gene therapy. An example of one such condition with well-studied genetic lesions that have been targeted with replacement gene therapy is muscular dystrophy.
Statistics for the prevalence of combined muscular dystrophies are scarce. A four state study estimated that 1 of every 5,600 to 7,700 males ages 5 through 24 years of age have either Duchenne or Becker muscular dystrophy, with 82% of such patients being wheelchair bound by 10 to 14 years of age and 90% by ages 15 to 24. In the absence of family history, Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) combined are generally diagnosed about 2.5 years from symptom onset which is generally noticed at an average age of 2.5 years and presents as proximal muscle weakness causing waddling gait, toe-walking, lordosis, frequent falls, and difficulty in standing up and climbing up stairs. Many patients also present with cognitive impairment including lower than expected IQ. Symptoms progress more rapidly in DMD than BMD and the course of BMD is generally more benign. DMD is a severe X-linked recessive, progressive muscle-wasting disease which alone affects about 1 in 3,500 boys. Patients with DMD generally do not survive past their late teens or early twenties. The primary cause of death in patients with DMD is respiratory failure, due to intercostal muscle weakness, and/or cardiac complications including cardiomyopathy.
Dystrophin-related muscular dystrophies, including DMD and BMD, are caused by mutations in the dystrophin gene, which encodes a 427-kDa β-spectrin/α-actinin protein family member protein, and/or mutations in genes encoding components of the dystrophin-associated protein complex. Various mutations in dystrophin-related muscular dystrophies result in complete absence of dystrophin protein (null mutation) as well as reduced levels of dystrophin protein, shortened dystrophin protein (deletion mutation), truncated dystrophin protein (premature stop codon mutation), tissue specific misexpression or reduced expression of dystrophin protein, reduced dystrophin-associated protein complex formation and combinations thereof. The human dystrophin gene is located on the X chromosome at position Xp21 and spans about 2.5 Mb of genomic sequence and is composed of 79 exons. The resulting 14-kb mRNA dystrophin transcript is expressed, through three independently regulated promoters, predominantly in skeletal and cardiac muscle with minor expression in the brain. While there is some correlation between severity of the genetic lesion causing a dystrophin-related muscular dystrophy and severity of the disease phenotype, some large deletions, e.g., in the dystrophin rod domain, appear to be uncorrelated resulting in relatively benign phenotypes.
These clinically mild yet dramatically shortened dystrophin mutant proteins have been the basis for the development of mini-dystrophin genes used in various dystrophin gene therapies. While dystrophin gene therapies, whether based on delivery of a mini-dystrophin gene or other dystrophin encoding nucleic acid, have shown promise in the laboratory and pre-clinical studies with muscular dystrophy animal models, human clinical studies have been met with significant setbacks, including dystrophin replacement gene specific immunity in treated DMD subjects.